Hello all researchers:
I have started working on a hypothetical protein.
In silico (SWISS-prot, Hhpred, I-TASSER) analysis shows its homology to following proteins:
1. Hydrolase (peptidase)
2. Restriction enzyme
3. Transferase
4. Heme-binding protein
These are the ones with highest scores and common among these servers.
Now how can i proceed, which experiments can i perform to check if protein actually has one of these functions?
Thanks in advance.
First you have to clone the DNA sequence that codes for your protein into a plasmid and then recombinantly express and purify your protein from cells. Using BL21(DE3) E.coli cells is typically the easiest. pET Plasmids are typically the easiest to work with also.
Then read about the enzymes capable of the functions. Buy the cheapest enzyme capable of the function and use it as a positive control. At this point, it doesn't matter what the kinetic parameters are for the enzymes. You just need to detect product or the depletion of substrate with the positive control enzyme indicating that the assay is working. After you get the positive control to work, then assay your recombinant enzyme of interest with the same assay using the same concentration of enzyme as you used for the positive control. Do the assay for longer than recommended just in case the activity is actually low. If you see formation of product or depletion of substrate then your enzyme has that function. If you don't see that then your enzyme doesn't have that function and the in-silico prediction software is wrong which happens fairly often.
In the case of a restriction enzyme assay, you can get an empty vector plasmid and it will probably be cut with BAMHI restriction enzyme. Make sure it has the restriction site by looking at the plasmid sequence in Benchling or SnapGene. If not use another appropriate enzyme. Do the cleavage assay, run the products on an agarose gel pre-stained with Ethidium bromide or another fluorescent DNA intercalating dye. For restriction enzyme assay use about 2 micrograms of plasmid because sensitivity of the assay is not as good as advertised. Incubate the plasmid to be cut with the restriction enzymes overnight at 37 celsius
Lane 1: DNA Ladder,
Lane 2: plasmid (2 micrograms) to be cut only without restriction enzymes
Lane 3: BaMHI restriction enzyme + plasmid to be cut (2 micrograms)
Lane 4: Recombinant enzyme of interest + plasmid to be cut (2 micrograms)
If lane 4 looks kind of like lane 3 but not like lane 2 then your enzyme is a restriction enzyme. If not then it is probably not a restriction enzyme or maybe you are just unlucky on the plasmid doesn't have the restriction site that this enzyme uses. To test that just do a DNA binding assay which just needs DNA and a stoichiometric excess of enzyme, no magnesium because magnesium usually is required for cutting DNA. Do you see a supershifted band? If yes then your enzyme binds DNA. If no then your enzyme doesn't. In such case, don't bother doing anymore DNA based assays.
For protease assays, casein is the usual protein substrate to be cleaved by the protease. So for positive control you could use Trypsin protease and you can compare that with your recombinant protein.
https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/elisa/protease-activity-assay
With respect to heme binding, is your protein abundantly expressed in blood? If not then why would it bind heme?
For any other assays, read the literature for the enzymes paying more attention to the methods section than to the abstract or introduction or results or discussion. Methods is how you do the assays. Replace your punitive enzyme with the enzyme they used as a positive control.
Adron Ung first of all, thank you so much for this detailed answer. It really provided me multiple ideas.
I have cloned a construct containing gene of interest with a FLAG tag to do protein analysis.
Regard heme binding: in I-TASSR, one of the homologs was heme-binding protein.
I am working on proteins in archaea not in humans.May be its somehow related to iron or else.
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